Molecules 2018, 23, 27
12 of 14
13C-NMR (100 MHz, MeOD-d4)
δ
169.9, 138.8, 135.4, 133.0, 129.3 (q, J = 31.1 Hz), 127.4, 125.5, 124.1
(q, J = 270.7 Hz), 120.0 (q, J = 5.4 Hz), 118.3, 56.4, 35.9 ppm. HRMS calculated for C20H14Cl2N2O2F6
(M − H)−: 498.0337. Found: 498.0338.
1
2-Allyl-N1,N3-bis(3-ethynylphenyl)malonamide (33): H-NMR (400 MHz, MeOD-d4)
δ 7.74 (s, 2H), 7.55 (d,
J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.20 (d, J = 7.6 Hz, 2H), 5.93–5.83 (m, 1H), 5.18 (d, J = 17.2 Hz,
1H), 5.08 (d, J = 10.4 Hz, 1H), 3.54 (t, J = 7.2 Hz, 1H), 3.47 (s, 2H), 2.77 (t, J = 7.2 Hz, 2H) ppm.
13C-NMR (100 MHz, MeOD-d4)
δ 169.9, 139.5, 135.5, 130.0, 129.0, 124.6, 124.2, 121.8, 118.2, 84.0, 78.8,
56.2, 36.3 ppm. HRMS calculated for C22H18N2O2S2 (M − H)−: 342.1368. Found: 342.1364.
1
2-Allyl-N1,N3-bis(3-ethylphenyl)malonamide (34): H-NMR (400 MHz, CDCl3-d1)
δ
8.83 (s, 2H), 7.37 (d,
J = 7.6 Hz, 2H), 7.36 (s, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.96 (d, J = 7.6 Hz, 2H), 5.87–5.79 (m, 1H), 5.18
(d, J = 16.8 Hz, 1H), 5.08 (d, J = 10.4 Hz, 1H), 3.39 (t, J = 7.2 Hz, 1H), 2.79 (t, J = 7.2 Hz, 2H), 2.61 (q,
J = 7.6 Hz, 4H), 1.20 (t, J = 7.6 Hz, 6H) ppm. HRMS calculated for C22H26N2O2 (M
Found: 350.2003.
−
H)−: 350.1994.
1
2-Allyl-N1,N3-bis(3,5-dichlorophenyl)malonamide (35): H-NMR (400 MHz, CDCl3-d1)
δ 8.86 (s, 2H), 7.50
(d, J = 1.6 Hz, 4H), 7.13 (t, J = 1.6 Hz, 2H), 5.81–5.73 (m, 1H), 5.20 (d, J = 17.2 Hz, 1H), 5.14 (d, J = 10.4 Hz,
1H), 3.35 (t, J = 7.2 Hz, 1H), 2.77 (t, J = 7.2 Hz, 2H) ppm. HRMS calculated for C18H14Cl4N2O2
(M − H)−: 429.9809. Found: 429.9818.
1
2-Benzyl-N1,N3-bis(4-chloro-3-(trifluoromethyl)phenyl)malonamide (36): H-NMR (400 MHz, MeOD-d4)
δ
7.98 (d, J = 2.0 Hz, 2H), 7.79 (dd, J = 8.8, 2.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.29–7.25 (m, 5H), 3.71 (s,
2H) ppm. 13C-NMR (100 MHz, MeOD-d4)
δ
167.3, 138.2, 135.4, 132.9, 131.9, 129.3 (q, J = 31.2 Hz), 129.2,
128.7, 128.3, 126.8, 124.1 (q, J = 270.9 Hz), 121.4 (q, J = 5.6 Hz), 74.1, 45.1 ppm. HRMS calculated for
C24H16Cl2N2O2F6 (M − H)−: 548.0493. Found: 548.0500.
1
2-Benzyl-N1,N3-bis(3,5-dichlorophenyl)malonamide (37): H-NMR (400 MHz, MeOD-d4)
δ 7.57 (s, 4H),
7.28 (s, 2H), 7.25–7.15 (m, 5H), 3.71 (t, J = 8.0 Hz, 1H), 3.32 (d, J = 8.0 Hz, 2H) ppm. HRMS calculated
for C22H16Cl4N2O2 (M − H)−: 479.9966. Found: 479.9960.
1
2-Benzyl-N1,N3-bis(4-nitro-3-(trifluoromethyl)phenyl)malonamide (38): H-NMR (400 MHz, MeOD-d4)
δ
8.19 (s, 2H), 8.02 (s, 4H), 7.32–7.17 (m, 5H), 3.38 (s, 2H) ppm. 13C-NMR (100 MHz, DMSO-d6)
δ 167.8,
143.1, 141.6, 138.2, 128.6, 128.3, 127.6, 126.4, 123.0, 122.7, 121.9 (q, J = 271.5 Hz), 117.5 (q, J = 5.6 Hz),
56.7, 34.3 ppm. HRMS calculated for C24H16N4O6F6 (M − H)−: 570.0974. Found: 570.0975.
1
2-Benzyl-N1,N3-bis(3,5-bis(trifluoromethyl)phenyl)malonamide (39): H-NMR (400 MHz, CDCl3-d1)
δ 9.30
(s, 2H), 7.91 (s, 4H), 7.66 (s, 2H), 7.17–7.06 (m, 5H), 3.57 (t, J = 8.0 Hz, 1H), 3.34 (d, J = 8.0 Hz, 2H) ppm.
13C-NMR (100 MHz, DMSO-d6)
J = 271.2 Hz), 119.1, 116.5, 56.7, 34.4 ppm. HRMS calculated for C26H16N2O2F12 (M
Found: 616.1027.
δ
167.7, 140.5, 138.3, 130.7 (q, J = 32.5 Hz), 128.7, 128.3, 126.5, 123.1 (q,
−
H)−: 616.1020.
4.3. Biological Assay
Biofilm eradication synergy assay. The interaction of 26 and antibiotics commonly used for
eradicating MRSA in the biofilm was evaluated with an assay modified from the minimal biofilm
eradication concentration (MBEC) assay [15] and the microdilution checkerboard assay. Briefly,
MRSA (ATCC 33591) grown overnight was suspended in cation-adjusted MH (CAMH) broth to
a concentration of 5
×
105 cfu/mL and aliquoted into the wells of a 96-well plate. Then, the plate was
◦
covered by a sterile peg lid and incubated at 37 C for 24 h. The biofilm formed on the pegs was rinsed
with sterile PBS twice to remove loosely adherent planktonic cells followed by exposing to 26 and test
antibiotic at two-fold escalating concentrations, ranging from 1 to 64 mg/L, in a new 96-well plate for
24 h. Next, the peg lid was rinsed with PBS twice, transferred to another 96-well plate with 200 µL
of CAMH in the wells, and sonicated by using a table sonicator for 5 min. After sonication, the peg
lid was replaced by a flat lid, and the 96-well plate was incubated at 37 ◦C for 24 h. The MBEC was